A recombinant hybrid protein as antigen for an anti-blood stage malaria vaccine: a study on the conservation of a protective component

Recently we have shown that two hybrid proteins expressed in Escherichia coli confer protective immunity to Aotus monkeys against an experimental Plasmodium falciparum infection (Knapp et al., 1992). Both hybrid proteins carry a sequence containing amino acids 631 to 764 of the serine stretch protein SERP (Knapp et al., 1989b). We have studied the diversity of this SERP region in field isolates of P. falciparum. Genomic DNA was extracted from the blood of six donors from different endemic areas of Brazil and West Africa. The SERP region encoding amino acids 630 to 781 was amplified by polymerase chain reaction (PCR) and sequenced. Only conserved amino acid substitutions in maximally two positions of the analyzed SERP fragment could be detected which supports the suitability of this SERP region as a component of anti-blood stage malaria vaccine.

Vaccine strategies against schistosomiasis

In this review the authors analyze the effector and regulatory mechanisms in the immune response to schistosomiasis. To study these mechanisms two animal models were used, mouse and rat. The mouse totaly permissive host like human, show prominent-T cell control in the acquisition of resistance. But other mechanisms like antibody mediated cytotoxity (ADCC) involving eosinophils and IgG antibodies described in humans, are observed in rats. Also in this animal, it is observed specific IgE antibody high production and blood and tisssue eosinophilia. Using the rat model and schistosomula as target, some ADCC features have emerged: the cellular population involved are bone marrow derived inflammatory cell (mononuclear phagocytes, eosinophils and platelets), interacting with IgE through IgE Fc receptors. Immunization has been attempted using the recombinant protein Sm28/GST. Protection has been observed in rodents with significant decrease of parasite fecundity and egg viability affecting the number, size and volume of liver egg granulomas. The association of praziquantel and immunization with with Sm28/GST increases the resistance to infection and decreases egg viability. The authors suggest the possibility of the stablishment of a future vaccine against Schistosoma mansoni.

Harmonization of research and control in schistosomiasis

I have been employed by several different organizations during over 30 years working on schistosomiasis, the majority spent in endemic areas of Caribean, South America, Africa and the Western Pacific. Much of the work is best classified as applied research but sometimes it strayed to the extremes of either public health control programmes or pure research. Over this period, there have been several significant research developments that have altered our whole approach to control. Ideally, research and control should complement each other but, in reality, they sometimes have conflicting objectives. Public health workers understandably wish to provide immediate, shot-term protection to the communities in their care, but research workers may, within ethical limits, reasonably want to observe untreated communities for extended periods in order to understand the underluing process of transmission, disease pathogenesis and immunity to help develop more effective control measures. An example of this situation has occured recently in Senegal where water development projects seem to have favoured the introduction and spreed of Schistosoma mansoni in the Senegal River Basin. I have been asked to be the scientific consultant to the newly formed ESPOIR programme, linking European research organizations and the Senegal Ministry of Health, to reconcile the conflictiong aims of public health workers, wishing to use whatever funds can be obtained for an immediate chemotherapy to try to eliminate the focus, at present confined to the vicinity of a relatively small, commercially run sugar irrigation scheme; and research workers who see a rare chance to study the development of immune mechanisms in a adults in a community not previously exposed to the infection. This information could prove invaluable in understanding the development of immunity and the pathogenesis of disease, leading eventually to the development of vaccines to revolutionise the future approach to schistosomiasis...

Human IgE responses to Schistosoma mansoni and resistance to reinfection

Schistosoma mansoni infected Kenyan patients were treated and the intensities of their reinfections were followed over the next two years. in addition, their pre- and six month post-treatment serum levels of IgG1-4, IgM, and IgE, specific for schistosoma, egg and adult worm, were measured in ELISA. No reinfection took place before six months post-treatment. Reinfection intensities varied with age; the younger children becoming reinfected at significantly higher intensities than older individuals. When antibody and reinfection levels were compared, only the six month post-treatment IgE response against adult worm correlated negatively with intensities of reinfection and, therefore, was predictive of resistance or immunity to reinfection. IgE and IgG specific Western Blots were carried out. The adult worm antigens recognized by IgE were restricted compared with the IgG responses of the same patients, although no individual antigen was uniquely recognized by the IgE isotype. A dominant 22 kDa antigen was recognized by most but not all high IgE responders. Patients with IgE responses against this antigen suffered significantly lower subsequent levels of reinfection, compared with non-responders. A monospecific rabbit antiserum against the 22KDa adult worm antigen showed that this antigen is specifically located in the tegument of the adult worm and of 'lung' and 'liver' stage schistosomula, but is absent from the early 'skin' schistosomula. It is possible that this antigen is a target for human IgE mediated immune effector mechanisms active against the post skin stage schistosomula and that this is boosted by the death of adult worms.

Host innate immune responses to microbial pathogens.

Sepsis is among the leading causes of death worldwide and its incidence is increasing. Defined as the host response to infection, sepsis is a clinical syndrome considered to be the expression of a dysregulated immune reaction induced by danger signals that may lead to organ failure and death. Remarkable progresses have been made in our understanding of the molecular basis of host defenses in recent years. The host defense response is initiated by innate immune sensors of danger signals designated under the collective name of pattern-recognition receptors. Members of the family of microbial sensors include the complement system, the Toll-like receptors, the nucleotide-binding oligomerization domainlike receptors, the RIG-I-like helicases and the C-type lectin receptors. Ligand-activated pattern-recognition receptors kick off a cascade of intracellular events resulting in the expression of co-stimulatory molecules and release of effector molecules playing a fundamental role in the initiation of the innate and adaptive immune responses. Fine tuning of proinflammatory and anti-inflammatory reactions is critical for keeping the innate immune response in check. Overwhelming or dysregulated responses induced by infectious stimuli may have dramatic consequences for the host as shown by the profound derangements observed in sepsis. Unfortunately, translational research approaches aimed at the development of therapies targeting newly identified innate immune pathways have not held their promises. Indeed, all recent clinical investigations of adjunctive anti-sepsis treatments had little, if any, impact on morbidity and all-cause mortality of sepsis. Dissecting the mechanisms underlying the transition from infection to sepsis is essential for solving the sepsis enigma. Important components of the puzzle have already been identified, but the hunt must go on in the laboratory and at the bedside.

Schistosomiasis vaccine development: approaches and prospects

Mounting evidence for acquired immunity to schistosomiasis in humans supports the case for immunological intervention. On the other hand, rapid reinfection poses a threat to younger age groups due to the slow maturation of natural resistance. However, rational approaches, based on advances in immunology and molecular biology, have substantially increased the odds of producing an effective vaccine. Since the parasite cannot replicate in the human host and serious morbidity generally occurs only after a relatively long period of heavy worm burden, complete protection against infection is not essential. The chances of success would increase if more than one of the various host/parasite interphases were targeted, for example reducing morbidity through decreased worm loads as well as through suppression of egg production. Several promising schistosome antigens have now reached an advanced phase of development and are currently undergoing independent confirmatory testing according to a standardized protocol. A few molecules are being contemplated for scaled-up production but, so far, only one has reached the stage of industrial manufacture and safety testing. Since schistosomiasis cannot realistically be controlled by a single approach, vaccination is envisaged to be implemented in conjunction with other means of control, notably chemotherapy.

Gastrointestinal immune responses in HIV infected subjects

The gut associated lymphoid tissue is responsible for specific responses to intestinal antigens. During HIV infection, mucosal immune deficiency may account for the gastrointestinal infections. In this review we describe the humoral and cellular mucosal immune responses in normal and HIV-infected subjects.

Immune responses of IL-5 transgenic mice to parasites and aeroallergens

Eosinophils have long been thought to be effectors of immunity to helminths but have also been implicated in the pathogenesis of asthma. Patterns of cytokine production in the host may influence the pathogenesis of these diseases by regulating the activities of eosinophils and other components of the immune response. Mice which constitutively over-express IL-5 have profound and life-long eosinophilia in a restricted number of tissues. Although eosinophils from IL-5 transgenics are functionally competent for a number of parameters considered to be important in inflammation, untreated animals are overtly normal and free of disease. In addition, the responses of these animals when exposed to aeroallergens and helminths present a number of apparent paradoxes. Eosinophil accumulation in tissues adjacent to major airways is rapid and extensive in transgenics exposed to the aeroallergen, but even after treatment with antigen over many months these mice show no evidence of respiratory distress or pathology. Helminth-infected IL-5 transgenics and their non-transgenic littermates develop similar inflammatory responses at mucosal sites and are comparable for a number of T cell and antibody responses, but they differ considerably in their ability to clear some parasite species. The life-cycle of Nippostrongylus brasiliensis is significantly inhibited in IL-5 transgenics, but that of Toxocara canis is not. Our results also suggest that eosinophilia and/or over-expression of IL-5 may actually impair host resistance to Schistosoma mansoni and Trichinella spiralis. The pathogenesis of diseases in which eosinophils are involved may therefore be more complex than previously thought.

Redundant Notch1 and Notch2 signaling is necessary for IFNγ secretion by T helper 1 cells during infection with Leishmania major.

The protective immune response to intracellular parasites involves in most cases the differentiation of IFNγ-secreting CD4(+) T helper (Th) 1 cells. Notch receptors regulate cell differentiation during development but their implication in the polarization of peripheral CD4(+) T helper 1 cells is not well understood. Of the four Notch receptors, only Notch1 (N1) and Notch2 (N2) are expressed on activated CD4(+) T cells. To investigate the role of Notch in Th1 cell differentiation following parasite infection, mice with T cell-specific gene ablation of N1, N2 or both (N1N2(ΔCD4Cre)) were infected with the protozoan parasite Leishmania major. N1N2(ΔCD4Cre) mice, on the C57BL/6 L. major-resistant genetic background, developed unhealing lesions and uncontrolled parasitemia. Susceptibility correlated with impaired secretion of IFNγ by draining lymph node CD4(+) T cells and increased secretion of the IL-5 and IL-13 Th2 cytokines. Mice with single inactivation of N1 or N2 in their T cells were resistant to infection and developed a protective Th1 immune response, showing that CD4(+) T cell expression of N1 or N2 is redundant in driving Th1 differentiation. Furthermore, we show that Notch signaling is required for the secretion of IFNγ by Th1 cells. This effect is independent of CSL/RBP-Jκ, the major effector of Notch receptors, since L. major-infected mice with a RBP-Jκ deletion in their T cells were able to develop IFNγ-secreting Th1 cells, kill parasites and heal their lesions. Collectively, we demonstrate here a crucial role for RBP-Jκ-independent Notch signaling in the differentiation of a functional Th1 immune response following L. major infection.

Experimental Infection of Culex (Culex) quinquefasciatus and Aedes (Stegomyia) aegypti with Wuchereria bancrofti

A study was conducted to determine the susceptibility of local strains of Culex quinquefasciatus and Aedes aegypti to infection with the strain of Wuchereria bancrofti that occurs in Maceió, State of Alagoas, Brazil. Cx. quinquefasciatus blood fed simultaneously on the same microfilariae carrier ingested more blood and 2-3x more microfilariae than Ae. aegypti. Survival rates of both species of insects living for 21 days after blood feeding on microfilaraemic patients were not significantly different from the survival rates of mosquitoes that blood fed on amicrofilaraemic individuals. W. bancrofti parasites underwent normal development in Cx. quinquefasciatus, with third stage larvae first being recorded on the 11th day post infection, and their numbers increasing thereafter. Development of filariae in Ae. aegypti did not proceed beyond the first larval stage, and there was a progressively increasing number of non-viable larvae with the passage of time. It is concluded that Ae. aegypti is not involved in the transmission of W. bancrofti in Maceió.

LACK-reactive CD4+ T cells require autocrine IL-2 to mediate susceptibility to Leishmania major.

Mice from most inbred strains are resistant to infection with Leishmania major whereas mice from BALB strains are highly susceptible. Resistance and susceptibility result from the development of Th1 or Th2 cells, respectively. In this report, we document an IL-2 mRNA burst, preceding the reported early IL-4 response, in draining lymph nodes of susceptible mice infected with L. major. Neutralization of IL-2 during the first days of infection redirected Th1 cell maturation and resistance to L. major, through interference with the rapid IL-4 transcription in Leishmania homolog of mammalian RACK1 (LACK)-reactive CD4(+) cells. A burst of IL-2 transcripts also occurred in infected C57BL/6 mice that do not mount an early IL-4 response. However, although the LACK protein induced IL-2 transcripts in susceptible mice, it failed to trigger this response in resistant C57BL/6 mice. Reconstitution experiments using C.B.-17 SCID mice and LACK-reactive CD4(+) T cells from IL-2(-/-) BALB/c mice showed that triggering of the early IL-4 response required autocrine IL-2. Thus, in C57BL/6 mice, the inability of LACK-reactive CD4(+) T cells to express early IL-4 mRNA transcription, important for disease progression, appears due to an incapacity of these cells to produce IL-2.

Partial Protection of Mice against Trypanosoma cruzi after Immunizing with the TcY 72 Antigenic Preparation

A 72 kDa Trypanosoma cruzi glycoprotein recognized by the 164C11 monoclonal antibody (IgM isotype) was purified by preparative electrophoresis. The antigenic preparation obtained, named TcY 72, was used to immunize C57Bl/10 mice. The following results were observed after immunization: (1) induction of higher titres of IgG than IgM antibodies, as evaluated by indirect immunofluorescence; (2) significant DTH after injection of epimastigotes in mice footpads; (3) peak parasitemia in immunized mice was significantly reduced and animals were negative by 13 days post-infection, although the mice still succumb to infection; (4) the phenotypic analysis of spleen cell populations showed a decrease in the CD4/CD8 ratio in immunized mice. Taken as a whole, these findings indicate that TcY 72 is immunogenic and potentially important for protective immunity.

Leishmania (Leishmania) major-infected rhesus macaques (Macaca mulatta) develop varying levels of resistance against homologous re-infections

Seven rhesus macaques were infected intradermally with 10(7) promastigotes of Leishmania (Leishmania) major. All monkeys developed a localized, ulcerative, self-healing nodular skin lesion at the site of inoculation of the parasite. Non-specific chronic inflammation and/or tuberculoid-type granulomatous reaction were the main histopathological manifestations of the disease. Serum Leishmania-specific antibodies (IgG and IgG1) were detected by ELISA in all infected animals; immunoblot analyses indicated that numerous antigens were recognized. A very high degree of variability was observed in the parasite-specific cell-mediated immune responses [as detected by measuring delayed-type hypersensitivity (DTH) reaction, in vitro lymphocyte proliferation, and gamma interferon (IFN-gamma) production] for individuals over time post challenge. From all the recovered monkeys (which showed resolution of the lesions after 11 weeks of infection), 57.2% (4/7) and 28.6% (2/7) animals remained susceptible to secondary and tertiary infections, respectively, but the disease severity was altered (i.e. lesion size was smaller and healed faster than in the primary infection). The remaining monkeys exhibited complete resistance (i.e. no lesion) to each rechallenge. Despite the inability to consistently detect correlates of cell-mediated immunity to Leishmania or correlation between resistance to challenge and DTH, lymphocyte transformation or IFN-gamma production, partial or complete acquired resistance was conferred by experimental infection. This primate model should be useful for measuring vaccine effectiveness against the human disease.

Neutrophil-derived CCL3 is essential for the rapid recruitment of dendritic cells to the site of Leishmania major inoculation in resistant mice.

Neutrophils are rapidly and massively recruited to sites of microbial infection, where they can influence the recruitment of dendritic cells. Here, we have analyzed the role of neutrophil released chemokines in the early recruitment of dendritic cells (DCs) in an experimental model of Leishmania major infection. We show in vitro, as well as during infection, that the parasite induced the expression of CCL3 selectively in neutrophils from L. major resistant mice. Neutrophil-secreted CCL3 was critical in chemotaxis of immature DCs, an effect lost upon CCL3 neutralisation. Depletion of neutrophils prior to infection, as well as pharmacological or genetic inhibition of CCL3, resulted in a significant decrease in DC recruitment at the site of parasite inoculation. Decreased DC recruitment in CCL3(-/-) mice was corrected by the transfer of wild type neutrophils at the time of infection. The early release of CCL3 by neutrophils was further shown to have a transient impact on the development of a protective immune response. Altogether, we identified a novel role for neutrophil-secreted CCL3 in the first wave of DC recruitment to the site of infection with L. major, suggesting that the selective release of neutrophil-secreted chemokines may regulate the development of immune response to pathogens.

The effect of treatment on the age-antibody relationship in children infected with Schistosoma mansoni and Schistosoma haematobium

The effect of praziquantel treatment on the age-antibody relationship was studied in 174 children aged between 6 and 17 years from a schistosome endemic area in Zimbabwe. The children were co-infected with Schistosoma mansoni and S. haematobium with infection prevalences of 74% and 53% respectively. Antibody levels for the isotypes IgA, IgE, IgM, IgG1, IgG2, IgG3 and IgG4, directed against soluble egg antigen were measured using an indirect ELISA assay. Treatment resulted in a significant increase in levels of IgG2 and IgG3 while levels of IgA decreased significantly. In untreated children there were significant decreases in levels of IgG4. Treatment also resulted in significant alteration in the age-antibody profiles for the isotypes IgE, IgM, IgG1 and IgG2 in treated children but not in untreated children. The results are discussed in the context of factors believed to give rise to the age-antibody relationship; i.e. age-related exposure patterns, age-related development of acquired immunity, age-related hormonal changes and age-related changes in innate susceptibility to infection.